Alternative Splicing and Expression of Class II Tubulin Beta (TUBB2B) Are Associated with Outcome in Diffuse Large B-Cell Lymphoma

The aim was to compare differentially expressed exons and splicing variants between diffuse large B-cell lymphoma (DLBCL) patients, who had relapsed or remained in remission after dose dense chemoimmunotherapy.

We performed genome-wide exon array analysis from four DLBCL cell lines and 38 tumor tissues from young (<65 years) DLBCL patients with high-risk (aaIPI>1) disease. The patients were treated in a Nordic phase II protocol with six courses of R-CHOEP-14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and high dose cytarabine. At the time of the analysis, median follow up was 34 months, predicted 3-year progression free survival (PFS) 78% and overall survival (OS) 79%. RNA for quantitative PCR validation was available from 20 patients. Two DLBCL cell lines and eight patient samples were further analyzed with high throughput RNA sequencing. In addition, microarray data set from 233 DLBCL patients treated with chemoimmunotherapy (Lymphoma/Leukemia Molecular Profiling Project (LLMPP)) was utilized for validation.

Differentially expressed exons between relapsed and non-relapsed patients were screened using criteria of p ≤ 0.05 and fold change ≥1.6 converting to 566 differentially expressed genes, of which 131 coded proteins. One of the identified genes with possible alternative splicing was TUBB2B, which encodes therapeutic target of taxanes and vinca alkaloids. The expression of TUBB2B, and specifically the expression of exon 3, was found to be suppressed in relapsed patients in comparison to patients remaining in remission. Differential expression of TUBB2B whole transcript and exon 3 was confirmed with RNAseq and quantitative PCR. Studies in lymphoma cell lines provided further support for the existence of different TUBB2B isoforms. According to Kaplan Meier estimates the patients with high (>median) expression levels of TUBB2B exon 3 had better 3-year PFS and lymphoma related OS rates than the patients with low expression levels (95% vs. 61%, p=0.015 for PFS, 100% vs. 75%, p=0.024 for OS). The prognostic significance of TUBB2B gene expression was validated in LLMPP data set (3-year OS 80% vs. 67%, p=0.040).

The results provide evidence that differential expression and splicing of TUBB2B gene can discriminate the outcome of homogenously treated high risk DLBCL patients.

No relevant conflicts of interest to declare.